KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Rat Angiotensin I Converting
Enzyme (ACE) ELISA
For the quantitative determination of rat ACE in serum, plasma, cell
culture supernatants, body fluid and tissue homogenate

Cat. No. KT-51021

For Research Use Only. Not for use in diagnostic procedures.

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Product Information
Rat Angiotensin I Converting Enzyme (ACE) ELISA
Cat. No. KT-51021

INTENDED USEThis ACE ELISA kit is a 1.5 hour solid-phase ELISA designed for the quantitative determination of Rat
ACE. This ELISA kit is for research use only, not for therapeutic or diagnostic applications!

PRINCIPLE
ACE ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-ACE
antibody and a ACE-HRP conjugate. The assay sample and buffer are incubated together with ACE-HRP
conjugate in pre-coated plate for one hour. After the incubation period, the wells are decanted andwashed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the
enzyme-substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop thereaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometricallyat 450 nm in a microplate reader. The intensity of the color is inversely proportional to the ACE
concentration since ACE from samples and ACE-HRP conjugate compete for the anti-ACE antibody
binding site. Since the number of sites is limited, as more sites are occupied by ACE from the sample,
fewer sites are left to bind ACE-HRP conjugate. A calibration curve is plotted relating the intensity of thecolor (O.D.) to the concentration of calibrators. The ACE concentration in each sample is interpolated
from this calibration curve.

COMPONENTS
Reagents

Quantity

Microtiter Plate

96 wells

Calibrator 1 (0 ng/mL)

1

Calibrator 2 (5 ng/mL)

1

Calibrator 3 (10 ng/mL)

1

Calibrator 4 (25 ng/mL)

1

Calibrator 5 (50 ng/mL)

1

Calibrator 6 (100 ng/mL)

1

Enzyme Conjugate

1 x 6 mL

Substrate A

1 x 6 mL

Substrate B

1 x 6 mL

Stop Solution

1 x 6 mL

Wash Buffer (100X concentrate)

1 x 10 mL

Balance Solution

1 x 3 mL

Note: The balance solution is used only when the sample is cell culture supernatants, body fluid andtissue homogenate; if the sample is serum or plasma, then the balance solution is a superfluous reagent.

STORAGEAll reagents provided are stored at 4°C. Refer to the expiration date on the label.
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KAMIYA BIOMEDICAL COMPANYSAMPLE COLLECTION AND STORAGE
Serum
Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at
4°C. Centrifuge at approximately 1,000 x g (or 3,000 rpm) for 15 mintues. Remove serum and assay
immediately or aliquot and store samples at -20°C or -80°C.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000 xg (or 3,000 rpm) at 4°C within 30 minutes of collection. Assay immediately or aliquot and store samples at-20°C or -80°C.

Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues wererinsed in ice-cold PBS (0.02 mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed before
homogenization. Minced the tissues to small pieces and homogenized them in a certain amount of PBS
with a glass homogenizer on ice. The resulting suspension was subjected to ultrasonication or to two
freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugatedfor 15 minutes at 1,500 x g (or 5,000 rpm). Remove the supernate and assay immediately or aliquot andstore samples at -20°C or -80°C.

Cell lysates
Cells should be lysed according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation. Suspension cells
can be collected by centrifugation directly.
2. Wash cells three times in PBS.
3. Cells were resuspended in PBS and subjected to ultrasonication for 3 times. Alternatively, freeze cells
at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.
4. Centrifuge at 1,000 x g (or 3,000 rpm) for 15 minutes at 4°C to remove cellular debris.
5. Assay immediately or store samples at -20°C or -80°C.

Cell culture supernatants and other body fluids
Centrifuge cell culture media at 1,000 x g (or 3,000 rpm) for 15 minutes to remove debris. Assay
immediately or store samples at -20°C or -80°C.
NOTE:
1. Samples should be aliquoted and must be stored at -20°C (less than 3 months) or -80°C (less than 6months) to avoid loss of bioactivity and contamination. If samples are to be run within 24 hours, they
may be stored at 4°C. Avoid repeated freeze-thaw cycles.
2. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently.
3. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA
results due to the impacts of certain chemicals.
4. Samples containing a visible precipitate must be clarified prior to use in the assay. Care should betaken to minimize hemolysis. Do not use grossly hemolyzed or lipemic specimens.
5. Do not use heat-treated specimens.

MATERIALS REQUIRED BUT NOT SUPPLIED
1. Precision pipettors and disposable tips to deliver 10-1,000 µL. A multi-channel pipette is desirable forlarge assays.
2. 100 mL and 1 liter graduated cylinders.
3. Distilled or deionized water.
4. Tubes to prepare sample dilutions.
5. Absorbent paper.
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6. Microplate reader capable of measuring absorbance at 450 nm.
7. Centrifuge capable of 3,000 x g.
8. Microplate washer or washing bottle.
9. Incubator (37°C).
10. Data analysis and graphing software.

SAMPLE PREPARATION
1. Kamiya Biomedical Company is only responsible for the kit itself, but not for the samples consumed
during the assay. The user should calculate the possible amount of the samples used in the wholetest. Please reserve sufficient amount of samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of thecalibration curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of
the kit is necessary.
4. Owing to the possibility of mismatching between antigen from other resource and antibody used in our
kits (e.g. antibody targets conformational epitope rather than linear epitope), some native or
recombinant proteins from other manufacturers may not be recognized by our products.
5. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell
culture supernatant may not be detected by the kit.
6. Fresh samples without long term storage is recommended for the test. Otherwise, protein
degradation and denaturalization may occur in those samples and finally lead to wrong results.

REAGENT PREPARATION
1. Bring all kit components and samples to room temperature (20-25°C) before use.
2. Samples - Please predict the concentration before assaying. If concentrations are unknown or notwithin the detection range, a preliminary experiment is recommended to determine the optimal
dilution. PBS (pH 7.0-7.2) or 0.9% physiological saline can be used as dilution buffer.
3. Wash Solution - Dilute 10 mL of Wash Solution concentrate (100x) with 990 mL of deionized or
distilled water to prepare 1,000 mL of Wash Solution (1x). If crystals have formed in the concentrate,
warm to room temperature and mix gently until the crystals have completely dissolved. The 1x wash
solution is stable for 2 weeks at 4°C.
4. Do not dilute the other components which are ready-to-use.

ASSAY PROCEDURE
Please read Reagent Preparation before starting assay procedure. It is recommended that all Calibrators
and Samples be added in duplicate to the microtiter plate. It is strongly recommended to do a preliminary
experiment before measuring all samples.
1. Secure the desired number of coated wells in the holder, then add 100 µL of Calibrators or Samples
to the appropriate well in the antibody pre-coated Microtiter Plate. Add 100 µL of PBS (pH 7.0-7.2) inthe blank control well.
2. Dispense 10 µL of Balance Solution into 100 µL specimens, mix well. (NOTE: This step is required
when the sample is cell culture supernatants, body fluid and tissue homogenate; if the sample isserum or plasma, then this step should be skipped.)
3. Add 50 µL of Conjugate to each well (NOT blank control well). Mix well. Mixing well in this step isimportant. Cover and incubate the plate for 1 hour at 37°C.
4. Wash the microtiter plate using one of the specified methods indicated below:
5. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper
waste container. Fill in each well completely with 1x wash solution, and then aspirate contents of theplate into a sink or proper waste container. Repeat this procedure five times for a total of FIVE
washes. After washing, invert plate, and blot dry by hitting the plate onto absorbent paper or paper
towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing theplate to assure that all strips remain securely in frame. Complete removal of liquid at each step isessential to good performance.
6. Automated Washing: Wash plate FIVE times with diluted wash solution (350-400 µL/well/wash) using
an auto washer. After washing, dry the plate as above. It is recommended that the washer be set fora soaking time of 10 seconds and shaking time of 5 seconds between each wash.
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7. Add 50 µL Substrate A and 50 µL Substrate B to each well including blank control well, subsequently.
Cover and incubate for 10-15 minutes at 37°C. (Avoid sunlight).
8. Add 50 µL of Stop Solution to each well including blank control well. Mix well.
9. Determine the Optical Density (O.D.) at 450 nm using a microplate reader immediately.

CALCULATION OF RESULTS
1. The calibration curve is used to determine the amount of samples.
2. First, average the duplicate readings for each calibrator and sample. All O.D. values are subtracted
by the mean value of blank control before result interpretation. DO NOT subtract the O.D. of
calibrator zero.
3. Construct a calibration curve by plotting the average O.D. for each calibrator on the horizontal (X) axis
against the concentration on the vertical (Y) axis, and draw a best fit curve using graph paper or
statistical software to generate a four parameter logistic (4-PL) curve-fit or logit-log linear regression
curve. An x-axis for the optical density and a y-axis for the concentration is also a choice. The data
may be linearized by plotting the log of the concentrations versus the log of the O.D. and the best fit
line can be determined by regression analysis.
4. Calculate the concentration of samples corresponding to the mean absorbance from the calibration
curve.
NOTE:
5. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit
age can cause variation in result. Each user should obtain their own calibration curve.
6. If samples have been diluted, the concentration read from calibration curve must be multiplied by the
dilution factor.
7. If specimens generate values higher than the highest calibrator, dilute the specimens and repeat theassay.

CERTIFICATE OF ANALYSIS
1.
2.
3.
4.

In the same lot CV%: <10
Different lot CV%: <10
Spike Recovery: 92-101%
Linearity

Range %
1:2
94 - 102
1:4
92 - 104
1:8
98 - 105
1:16
94 - 109
5. Sensitivity: The sensitivity in this assay is 1.0 ng/mL.
6. Specificity: This assay has high sensitivity and excellent specificity for detection of ACE. No
significant cross-reactivity or interference between ACE and analogues was observed.
NOTE: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity
detection between ACE and all the analogues, therefore, cross reaction may still exist in somecases.

SAFETY NOTES
1. This kit contains small amount of 3,3',5,5'-Tetramethylbenzidine (TMB) in Substrate B. TMB is noncarcinogenic but it is hazardous in case of skin contact, eye contact, ingestion and inhalation. In caseof contact, rinse affected area with plenty of water.
2. The Stop Solution provided with this kit is an acid solution. Wear protective gloves, clothing, and faceprotection.
3. Care should be taken when handling the Calibrator because of the known and unknown effects of it.
4. Care should also be taken to avoid contact of skin or eyes with other kit reagents or specimens. In thecase of contact, wash immediately with water.
5. Do not pipette by mouth.
6. Avoid generation of aerosols.
7. Waste must be disposed of in accordance with federal, state and local environmental control
regulations.
8. All blood components and biological materials should be handled as potentially hazardous.
Decontaminate and dispose specimens and all potentially contaminated materials as they could
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contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1hour at 121.5°C.

QUALITY CONTROL
1. It is recommended that all calibrators, controls and samples be run in duplicate. Calibrators andsamples must be assayed at the same time.
2. The coefficient of determination of the calibration curve should be ≥0.95.
3. Cover or cap all kit components and store at 4°C when not in use.
4. Microtiter plates should be allowed to come to room temperature before opening the foil bags. Oncethe desired number of strips has been removed, immediately reseal the bag with desiccants and store
at 4°C to maintain plate integrity.
5. Samples should be collected in pyrogen/endotoxin-free tubes.
6. Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles offrozen samples. Thaw completely and mix well prior to analysis.
7. When possible, avoid use of badly hemolyzed or lipemic serum. If large amounts of particulate matterare present, centrifuge or filter prior to analysis.
8. When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal
incubation times for all wells.
9. Do not mix or interchange different reagent lots from various kit lots.
10. Do not use reagents after the kit expiration date.
11. Read absorbance immediately after adding the stop solution.
12. Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash
Solution provided. All residual wash liquid must be drained from the wells by efficient aspiration or bydecantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper
directly into the wells.
13. Because TMB is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB
and metal, otherwise color may develop.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068 Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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